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Abstract

Background

HIV resistance affects virological response to therapy and efficacy of prophylaxis
in mother-to-child-transmission. The study aims to assess the prevalence of HIV primary
resistance in pregnant women naïve to antiretrovirals.

Methods

Cross sectional baseline analysis of a cohort of HIV + pregnant women (HPW) enrolled
in the study entitled Antiretroviral Management of Antenatal and Natal HIV Infection
(AMANI, peace in Kiswahili language). The AMANI study began in May 2010 in Dodoma,
Tanzania. In this observational cohort, antiretroviral treatment was provided to all
women from the 28th week of gestation until the end of the breastfeeding period. Baseline CD4 cell count,
viral load and HIV drug-resistance genotype were collected.

Results

Drug-resistance analysis was performed on 97 naïve infected-mothers. The prevalence
of all primary drug resistance and primary non-nucleoside reverse-transcriptase inhibitors
resistance was 11.9% and 7.5%, respectively. K103S was found in two women with no
M184V detection. HIV-1 subtype A was the most commonly identified, with a high prevalence
of subtype A1, followed by C, D, C/D recombinant, A/C recombinant and A/D recombinant.
HIV drug- resistance mutations were detected in A1 and C subtypes.

Conclusion

Our study reports an 11.9% prevalence rate of primary drug resistance in naïve HIV-infected
pregnant women from a remote area of Tanzania. Considering that the non-nucleoside
reverse-transcriptase inhibitors are part of the first-line antiretroviral regimen
in Tanzania and all of Africa, resistance surveys should be prioritized in settings
where antiretroviral therapy programs are scaled up.

Keywords:

HIV-drug resistance; MTCT; HIV-genotype; Low-resources countries

Background

In 2010, 5 million people had access to HIV treatment in Sub-Saharan Africa, about
49% of those in need. In Eastern and Southern Africa, 56% of eligible patients had
access to therapy, versus 10% treated in 2009 [1]. The scale-up of HIV treatment in low- and middle-income countries has been crucial
to substantially reduce AIDS-related morbidity and mortality as well as mother-to-child-transmission
(MTCT). As access to antiretroviral therapy expands, HIV drug resistance (HIVDR) inevitably
emerges because of HIV’s high mutation rate, viral recombination, and the patient’s
need for sustained, lifelong treatment. The probability of drug resistance escalation
during treatment has been estimated at 27% every 6 years [2]. The HIVDR insurgence may be related to a different mechanism. It could be due to
drug pressure in patients receiving antiretroviral therapy (ART) because of suboptimal
adherence, pharmacodynamic factors, or use of inadequate or suboptimal regimes. In
recently infected individuals, HIVDR may be transmitted from one individual to another.
Finally, HIVDR can be transmitted or acquired in individuals with chronic infections.

The implications of transmitted drug resistance are a cause of concern for the scaling
up of HIV programs, as HIV resistance also affects the efficacy of MTCT prophylaxis.

Drug sensitivity testing (DST) is the standard of care in industrialized countries,
but is rarely available in resource-limited settings due to high costs and stringent
requirements for storage and transport of plasma. With the introduction of antiretroviral
drugs in low-resource countries (known for the largest assortment of non-B subtypes),
gaining a better understanding of the responsiveness to antiretroviral therapy and
HIV-1 drug resistance in non-B strains has become a priority. In such settings, patients
who do not respond to therapy are often blindly switched from a non-nucleoside reverse
transcriptase inhibitor (NNRTI) to a protease inhibitor (PI)-based regimen. However,
since treatment failure is detected late in most patients (at a stage when widespread
resistance is common), the risk of switching to regimens with limited efficacy increases.

Among determinant factors driving the emergence of HIVDR, virus- related factors play
a crucial role in the susceptibility to drug- resistance mutations. Viral subtypes
other than B share different ARV susceptibilities compared to HIV-1 non-B subtypes,
which are naturally more or less susceptible to specific drugs. Different results
were obtained in several studies that have compared the prevalence of drug-related
mutations in different HIV-1 non-B subtypes [3]: the recombinant form CRF02_AG is reported to be more susceptible to nelfinavir (NFV)
and ritonavir (RTV) than C and F subtypes; G subtype is more sensitive to tipranavir
(TPV) and lopinavir (LPV) than other subtypes [4], and C subtype has a greater risk of developing resistance to tenofovir (TDF) [5]. In a Ugandan study [6], the K103N mutation was relatively more frequent in C subtype- infected women failing
NNRTI-based therapy than in both A and D subtypes. The G190A/S mutation was considered
a common polymorphism in Israeli C subtype patients, but not in Indian C subtype individuals
[7,8]. Despite the variability of non-B HIV-1 subtypes in viral mutational patterns and
in vitro susceptibility, the benefit of treatment programs clearly outweigh the risks of emerging
HIV DR [3-8].

Future clinical studies designed to provide clinical and virological data in non-B
strains are of great interest. Additional information on the prevalence of drug-resistance
mutations in naïve HIV populations could be crucial for tailoring combination regimens.
Furthermore, it could help clinicians to decide whether DST prescription is necessary
before initiating therapy.

This study aims to assess the prevalence of HIV drug-related resistance and the circulation
of non-B subtype in pregnant women naïve to antiretrovirals in Dodoma region, central
mainland Tanzania.

Methods

Study design

The data provided are part of a nested case-control study of HIV resistance outcome
among the HIV + pregnant women (HPW) enrolled in the study entitled Antiretroviral
Management of Antenatal and Natal HIV Infection (AMANI, peace in Kiswahili language).
The AMANI study is an interventional study which aims to assess the feasibility of
ART use for preventing MTCT in a cohort of HIV-infected pregnant women. HAART is provided
to all women starting at the 28th week of gestation until the end of the breastfeeding period, within an integrated
MTCT prevention program. A systematic screening during a formal interview on previous
ART use including single-dose NVP is performed. Baseline CD4 cell count, viral load,
and HIV drug- resistance genotypes are collected at baseline, during pregnancy and
lactation.

The current study analyzed a subgroup of 97 pregnant women naïve to any antiretroviral
treatment. In order to be certain that there was no previous exposure to any ARV,
women were included in the study only if the first HIV positivity was discovered during
the current pregnancy. The AMANI study was approved by the Italian Ethical Board of
the “L. Spallanzani” National Institute for Infectious Diseases in November 2009 and
by the Tanzanian Medical Research Coordinating Committee of the National Institute
of Medical Research (NIMR), with certificate no. NIMR/HQ/R.8a/Vol.IX/907 in December
2009. All recruited women provided written informed consent.

HIV sequencing

HIV genotype analysis was performed on plasma samples by using a commercially available
HIV genotyping kit (ViroSeq HIV-1 Genotyping System version 2.0, Abbott Molecular).

In brief, RNA was extracted using a commercially available kit (QIAamp RNA Viral Mini
kit, Qiagen), retrotranscribed by murine leukaemia virus RT, and amplified with ampliTaq
Gold polymerase enzyme. Pol amplified products (containing the entire Protease (99-aa)
and the first 320 amino acids of the Reverse Transcriptase) were full-length sequenced
in sense and antisense orientations, using seven different overlapping sequence-specific
primers by an automated sequencer (ABI 3130, Applied Biosystems, Foster City, CA,
USA) [9,10]. Sequence data were analyzed by a specific HIV genotyping system software that automatically
assembles the seven sequence segments into a consensus sequence, which is then compared
to a B reference strain. Sequences having a mixture of wild-type and mutant residues
at single positions were considered to have a mutation at that position. When the
mixture was between two different mutations, both mutations were considered and reported.
To classify and identify polymorphisms and mutations associated with resistance to
ARVs, the FASTA sequences of the PR and RT were analyzed using the freely available
SDRM-2009 algorithm available in the Calibrated Population Resistance tool (CPR),
version 6.0 beta (http://cpr.stanford.edu/cpr.cgiwebcite). The SDRM algorithm (the SDRM worksheet shows all of the mutations present on the
ANRS, HIVdb, IAS-USA, Los Alamos, and Rega algorithm lists) [11] was applied to determine the prevalence of primary ARV- resistance mutations, using
a list of drug-resistance mutations that provide an estimate of resistance transmission
according to the WHO guidelines (http://hivdb.stanford.edu/cgi-bin/AgMutPrev.cgiwebcite).

Genotypic sub typing

Pol subtype was determined using phylogenetic analysis on HIV-1 pol-sequences. Briefly,
the sequences were aligned with HIV-1 reference sequences of all subtypes (http://www.hiv.lanl.govwebcite). The alignment was edited using the BioEdit program version 7.0.5.3. The phylogenetic
analysis of pol aligned sequences was performed by the maximum-likelihood method of MEGA version
5.05. The transversion model (GTR + I + G) of nucleotide substitution was chosen using
the Kimura two-parameter model as the best-fitting evolution model for tree reconstruction.
The reliability of the branching patterns was evaluated by bootstrapping (1,000 replicates).
Only bootstrap values >70% were evaluated. Subtype classification was also confirmed
by REGA subtype tool (http://www.bioafrica.net/rega-genotype/html/subtypinghiv.htmlwebcite) and COMET subtype tool (http://comet.retrovirology.lu/webcite). To improve the accuracy of subtype recombinant forms and unique forms, RDP3 software
(http://web.cbio.uct.ac.za/~darren/rdp.htmlwebcite) and Splits Tree software (http://www.splitstree.org/webcite) were used.

Statistical analysis

Descriptive statistics were used to analyze the epidemiological data. Chi- square
tests were used to assess differences between groups with reference to the occurrence
of drug-resistance mutations. A univariate analysis was performed to examine possible
demographic, clinical and viro-immunological factors related to the occurrence of
drug-resistance mutations. The Chi-square test was used for the categorical variable.
The T-test was used for comparison of the means for the quantitative variable; Wilcoxon
signed rank was used for comparison of medians. The significant level was set at 0.05.
All the analyses were performed using SPSS for Windows 12.0 (SPSS Inc, Chicago, Illinois
60606, USA).

Results

The enrollment phase of the AMANI study started on May 2010 at the Makole Urban Health
Centre (Makole UHC) in the municipality of Dodoma (Figure 1). During the nineteen months, 4,138 pregnant women attended the Ante Natal Clinic
(ANC), and 326 (7.8%) of these were found to be affected by HIV infection. Among them,
103 (31.6%) HPW were not eligible for the study (i.e. attending the ANC with gestational
age >28 weeks), 3 (1%) refused to participate (due to fear of drug side effects),
and 220 (67.5%) were included in the study. Thirty-six of them (16.4%) are currently
under evaluation for inclusion in the study. Twenty-seven (14.7%) of the remaining
184 (83.6% of 220) did not return after the enrollment visit and 12 (6,5%) dropped
out. Of the remaining 145 (78.8%) patients, 33 (22.8%) were already on HAART at enrollment.
Among the 112 (77.2%) HPWs not on HAART at baseline, 85 (75.9%) started ART for prevention
of MTCT at the 28th week of gestation, and 27 (24.1%) were eligible for therapy and started HAART (Figure 1).

Of the 112 patients originally not on HAART, 97 mothers with a first HIV positivity
during the current pregnancy and with no reported previous ART use were tested for
drug resistance before starting any antiretroviral drug (Table 1): 53 (54.6%) and 27 (27.8%) were defined as WHO stage 1 and 2, respectively. Unprotected
heterosexual intercourse was the risk factor for HIV infection reported by all HPWs
in the study. At baseline, the median CD4 count was 392 (IQR, 260-528) cells/mm3 and
Median log10 HIV RNA copies/mL was 4.80 (IQR, 4.03-9.28). The HIV genotypic drug-resistance assay
provided valuable results in 67 cases (69.0%). The overall prevalence of primary drug
resistance was 11.9% (8/67; 95% CI 0.04-0.20); the prevalence of primary drug class-specific
resistance was 1.5% (1/67; 0.01-0.04) for nucleoside reverse-transcriptase inhibitors
(NRTIs) and 7.5% (5/67; 0.01-0.14) for NNRTIs. K103S and M41L were found in two and
one women, respectively, while M184V was not detected. Interestingly, L89M protease
polymorphism (potentially associated with resistance to fosamprenavir, and to a lesser
extent to darunavir and lopinavir) was commonly detected with a 76.1% prevalence,
equally distributed in all different non-B clades.

K103N and M41L mutations were on five lists and the K103S mutation was on four lists.

The HIV-1 A subtype was the most commonly identified (36/67, 53.7%) with a high prevalence
of A1 subtype (31/67, 47%), followed by C (14/67, 21%), and D (9/67, 13%). Furthermore,
some circulating recombinant forms (CRFs): CRF10_CD (8/67, 12%), CRF35_AD recombinant
(1/67, 1%) and some unique recombinant forms (URFs) A1/C recombinant (4/67, 6%) were
reported. The phylogenetic tree appears to show a cluster of related infections in
this geographical area, particularly in A1 and C subtypes (Figure 2). Finally, two K103S drug-related mutations and one K103N polymorphism were all clustered
in the A1 subtype. Pregnant women with HIV viral strains harboring drug-resistance
mutations before any antiretroviral treatment did not significantly differ from women
with wild-type HIV in terms of demographic, clinical, virological and immunological
parameters (Table 2).

Discussion

The recent HIV drug resistance report, released by WHO in November 2012 [12], reports the available data on the estimated prevalence of HIVTDR between 2003 and
2010 in 72 surveyed areas. WHO recommends a minimum-resource method to assess HIVTDR
in resource-limited countries where transmitted HIV drug resistance is likely to be
seen first (such as in urban areas). Of the 72 surveys, 52 (72.2%) had a low prevalence
of resistance to all drug classes and 20 (27.8%) had a moderate prevalence classification
of resistance to ≥1 antiretroviral drug class). The first reports from Tanzania in
2005 showed a 4% and 9% NNRTI resistance in naïve populations from the Kagera-Kilimanjaro
regions and Dar-es-Salaam urban area, respectively [13,14]. More recently, in 2011, a 14.7% HIVDR prevalence in a naïve population was reported
in Mwanza [15]. Authors combined drug-resistance prevalence data obtained from both peripheral blood
mononuclear cells (PBMC) and plasma, whereas only 4 samples (9.5%) resulted positive
at bulk sequencing assay from plasma [15]. The NNRTI prevalence rates observed in these surveys are slightly higher than those
observed in previous reports in other eastern and western African countries.

Reports showed the effect of a proper timing for introduction of ART, as a proxy for
the amount of circulating drug-resistance HIV-1 strains at the population level, and
level of primary resistance [16]. The overall sample-weighted drug resistance prevalence was 5.6% (139 of 2436; 95%
CI 4.6–6.7), ranging from 1.1% (two of 176; 0.0–2.7) in Pretoria, South Africa, to
12.3% (22 of 179; 7.5–17.1) in Kampala, Uganda [16].

Regarding PI mutations, no major resistance mutations were observed in our study.
Nevertheless, the 76.1% prevalence of the L89M protease polymorphism raises concern.
Some amino acid polymorphisms occur at sites that have been associated with drug resistance
in the B-subtype virus [9]. The L89M mutation increases the catalytic efficiency and vitality of the HIV-1 protease
gene in the presence of other protease mutations in non-B African viral subtypes [17] and can determine a low accumulation of primary protease mutations in non-B subtypes
[18,19]. These findings suggest that in addition to the primary drug-related mutations already
described in B clades, particular attention should be paid to some natural polymorphisms
in the therapeutic management of patients infected by HIV-1 non-B subtypes.Our study
aimed to determine the prevalence of transmitted HIV drug-resistance mutations among
untreated patients and also provided novel data on the HIV-1 variants that circulate
in Tanzania. We confirmed previous results that reported a high genetic diversity
in the number of co-circulating variants with the predominance of A clade (53.7%),
and a high prevalence of the A1 subtype (47%). Different from a 2004 report that described
a low detection of drug resistance in A subtype compared to D subtype [20], we reported that the primary NNRTI drug-related mutations were all clustered in
the A1 subtype. This variant was described as one of the most prevalent variants among
young adults in Tanzania, Dar-es-Salaam [14] and adults in the Kilimangiaro, Kagera and Mwanza regions [13,15]. Thus, the implementation of a surveillance study on the molecular epidemiology of
different HIV strains appears strictly complementary to the data obtained from prevalence
studies of drug-related mutations using bulk sequencing.

Several limitations were encountered in our study. Despite systematic screening of
previous ART use, the mean number of pregnancies is three and the risk of unreported
ART use in the AMANI cohort may be significant. Nevertheless, most HIVDR mutations
were reported in parous women; the AMANI study was conducted in a single area; a single
population was targeted, and the estimates of drug resistance have wide CIs. However,
despite all these considerations, the 11.9% prevalence of drug resistance in a naïve
population where the first-line antiretroviral regimen is still based on a NNRTI-based
HAART raises concern. Further work should be done to determine if resistance is a
consequence of short-term exposure during pregnancy or if in fact these individuals
had already accessed ARVs. Together, this information could be used to guide the development
of ART policy guidelines in Tanzania. Against this background, the increasing rates
of antiretroviral resistance in adults and children from low-income settings represent
a potential threat and urgent actions are needed. First, human and resource efforts
should be doubled to deploy proven effective preventive methods. Second, early and
sustained ART use for preventing MTCT must be fully embraced [21] and the recent 2012 WHO programmatic update on HIV PMTC transmission [22] should be strongly supported to provide the option B-plus, the use of a life-long
triple therapy for the pregnant women.

Conclusion

Despite all the discussed considerations, the 11.9% prevalence of drug resistance
in a naïve population where the first-line antiretroviral regimen is still based on
a NNRTI-based HAART raises concern. Further work should be done to determine if resistance
is a consequence of short-term exposure during pregnancy or if in fact these individuals
had already accessed ARVs. Together, this information could be used to guide the development
of ART policy guidelines in Tanzania. Against this background, the increasing rates
of antiretroviral resistance in adults and children from low-income settings represent
a potential threat and urgent actions are needed. First, human and resource efforts
should be doubled to deploy proven effective preventive methods. Second, early and
sustained ART use for preventing MTCT must be fully embraced [21] and the recent 2012 WHO programmatic update on HIV PMTC transmission [22] should be strongly supported to provide the option B-plus, the use of a life-long
triple therapy for the pregnant women.

Competing interests

The authors declare that they have no conflict of interest.

Authors’ contributions

FV, EN, NB, GL and GI designed and supervised the study. BN and ZC carried out the
patient enrollment. CS and AC supervised the laboratory diagnostic analysis at DRH,
Tanzania. FF, AA, LF, CFP and MRC performed the HIV-Genotype analysis at INMI. PDN,
EN, FV and AC analyzed data and contributed to the preparation of the manuscript.
All authors read and approved the final manuscript.